The goal of this application is to determine the specific roles of individual caspase family members in T-cell activation and proliferation. We will use novel caspase-specific cell-permeable inhibitors and activity-based probes, which we recently designed and characterized, to probe caspase function. Caspases are a family of cysteine-aspartyl proteases that are essential for apoptosis, pyroptosis, and inflammation. Humans have 11 caspase isoforms where initiator caspases-2, -8, -9 and -10 and executioner caspases-3, -6, and -7 are responsible for the induction of apoptosis and cellular dismantling, respectively. Exciting new data suggest that several of these apoptosis-associated caspases have unexpected non-canonical functions in cellular differentiation and proliferation, neuronal pruning, and synaptic plasticity. However, very few details about the roles and contributions of distinct caspases to these biological phenomena have been forthcoming. The lack of mechanistic insights is primarily due to the close structural homology and overlapping substrate specificity among the caspase family members. Another limitation is the high promiscuity of the currently available library of caspase peptide-based inhibitors, substrates, and probes. Therefore, the current set of caspase-targeted molecules does not provide adequate resolution to precisely assign biological roles to individual caspases in a complex cellular environment. We recently developed a unique approach that incorporates unnatural amino acids into the canonical caspase-recognition peptides and optimized the reactivity of chemical leaving groups to ensure our peptide-based molecules are specifically targeted to individual caspases. We have amassed sufficient preliminary data to identify first-in-class caspase-3 and caspase-8 specific peptide probes, thereby targeting a critical executioner and an initiator caspase, respectively. Here, we will apply cell-permeable versions of our designed molecules to investigate the non-canonical functions of caspases-3 and -8 in human T-cell activation. During apoptosis, caspases cleave nuclear proteins involved in transcription and translation and we hypothesize that a subset of these substrates are selectively targeted by caspases-3 and/or -8 during T-cell expansion. Therefore, the proteolytic activity of caspases-3 and -8 will significantl impact the transcription and translation of many key cellular proteins that determine T-cell function. We propose to use mass spectrometry-based proteomics to elucidate the cellular substrates of caspases-3 and -8 and RNA-Seq transcriptomics to shed light on downstream translational and transcriptional consequences on T-cell reprogramming. Our efforts will generate a universally applicable set of tools that can be extended to probe the functions of individual caspases during other biological processes, including stem cell differentiation, erythropoiesis, and cell migration as well as produce a paradigm shift in how caspases control non-apoptotic biological processes in health and disease.